Fgm 2 singlequots

Normal and diseased lung fibroblast were maintained in fibroblast growth medium (Lonza, Cat# CC-3132) supplement with the FGM-2 SingleQuots supplement (Lonza, Cat# CC-4126) in 10 cm dishes. For the ¹³C-glycogen enrichment experiment, fibroblast cells were allowed to reach ~50% confluency, followed by the addition of DMEM base media supplemented with 10 mM ¹³C₆-glucose, 2 mM Gln, 10% dialyzed fetal bovine serum in a CO₂ incubator maintained at 37 °C. At the designated timepoints, cells were washed with cold PBS three times followed by extraction with 50% methanol and separated into polar, insoluble fraction that contains the glycogen. For the ¹³C-glycogen washout experiment, fibroblast cells were grown in ¹³C₆-glucose enrichment media for 48 h followed by the replacement of DMEM base media supplement with ¹²C-glucose. At designated timepoints, cells were washed with cold PBS three times followed by extraction with 50% methanol and separated into polar and the glycogen-containing insoluble fraction.

Vendor and donor details for the cell types used in this work are provided in Table 1. All cells were expanded according to their respective manufacturer’s protocols to establish baseline frozen stocks; this ensured consistent procedures and passage numbers across experiments. Primary human retinal microvascular EC were purchased from Angio-Proteomie (Boston, MA) and expanded in the manufacturer’s Endothelial Growth Medium. This EC source was used for all co-culture studies conducted. Primary human dermal fibroblasts and normal human lung fibroblasts were purchased from Lonza (Basel, Switzerland) and expanded in the manufacturer’s recommended medium, Fibroblast Growth Medium-2 (FGM-2, catalog # CC-3132; comprised of FBM™ Basal Medium and FGM™-2 SingleQuots™ supplements). Immortalized human retinal pericytes were generated by Pfizer from primary cells acquired from Angio-Proteomie and expanded in Angio-Proteomie’s Pericyte Growth Medium.
For the experiments described in this study, cells were thawed into flasks with either EGM-2MV (Lonza, catalog # CC-3202) for EC or FGM-2 for fibroblasts and pericytes. Media was changed the next day. Cells typically reached 70%–80% confluence within 2–4 days and were harvested for seeding in PREDICT96 plates as described below.

Cancer-associated fibroblasts were differentiated from normal human dermal fibroblasts (Lonza) using four melanoma cell lines: A375 and Hs294T obtained from the American Type Culture Collection and WM1341D and WM9 cell lines purchased from Rockland Immunochemicals, Inc. Fibroblasts were cultured in FBM (Fibroblast Growth Basal Medium, Lonza) cell culture medium (supplemented with FGM™-2 SingleQuots™ from Lonza), whereas melanoma cells were grown in DMEM (Dulbecco’s Modified Eagle Medium) medium containing 4.5 g/l glucose and 1.5 g/l NaHCO₃ supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and antibiotics (10,000 U/ml penicillin, 10 mg/ml streptomycin, 25 µg/ml amphotericin B). Cells were cultured in 25 cm² tissue culture flasks (VWR) at 37 °C in 5%CO₂/95% humidified air and passaged twice a week using 0.25% trypsin/0.05% EDTA solution (IITD PAN, Wroclaw, Poland).